We have invented a new method for making valproic acid derivatives. The valproic acid derivatives can be used to make immunoreactive valproic acid conjugates.
Valproic acid is a low molecular weight, saturated fatty acid with significant utility as an antiepileptic drug. Valproic acid is used to treat a variety of convulsant disorders, and is especially effective in the treatment of petit mal absence, generalized tonic-clonic seizures, and myoclonic disorders. Valproic acid administration has also resulted in substantial improvement of patients with Lennox syndrome, which is extremely resistant to almost all drug treatment. Significantly, unlike many other anticonvulsant drugs, a development of tolerance by a patient to the anticonvulsant effects of valproic acid does not typically occur. A lack of tolerance development means that drug efficacy can be maintained for a given level of administration. The optimal therapeutic range of valproic acid depends on a number of factors, including the method of administration, and the particular convulsant condition being treated, but generally lies between about 50 .mu.g and about 100 .mu.g per mL of serum.
The wide acceptance and medical use of valproic acid, combined with a low and narrow optimal therapeutic serum concentration, necessitates a fast and accurate procedure for monitoring the level of valproic acid in a patient's physiological fluids. Monitoring of a patient's serum valproic acid level can be critical to medical decision-making. Furthermore, a hospital or clinical environment can generate a substantial number of physiological fluid samples obtained from patients receiving valproic acid. Hence, a method to detect and quantify valproic acid levels in multiple test samples quickly and accurately is highly desirable.
Methods for detecting valproic acid in physiological fluids are limited in part because of the small size of the valproic acid molecule and because valproic acid shows essentially no absorption characteristics in the ultraviolet spectrum. Known methods for detecting valproic acid include gas-liquid chromatography, high pressure liquid chromatography and mass spectrometry. These methods require expensive instrumentation, extensive test sample preparation, and lengthly technician training. All these factors can impede and delay analysis of a test sample for valproic acid. Additionally, these methods are not suitable for the rapid and accurate analysis of the large number of patient test samples that can be generated by the valproic acid monitoring programs of many hospitals and convulsant treatment clinics. Hence, a simple, fast, and efficient method of detecting and quantifying valproic acid in multiple physiological fluids samples has been pursued.
Immunological assay methods have shown considerable utility for the detection and quantification of valproic acid in test samples. An immunoassay can be based upon the detection of an immunoprecipitation reaction. An immunoprecipitation reaction can occur when two reaction partners, each with a specific binding affinity for the other, are combined in a suitable liquid medium. The reaction partners can be an antigen and a specific binding partner for the antigen, such as an antibody. Once initiated in a liquid medium, the immunoprecipitation reaction results in the formation of immunoprecipitates, or antibody-antigen complexes in the liquid medium.
The presence of immunoprecipitates in the liquid medium can change optical properties, such as light scattering and light absorption properties of the liquid medium, due to attenuation of incident light energy by the immunoprecipitates. These changes can be detected by an appropriate photometer in a photometric immunoassay. Photometric immunoassay techniques include both nephelometric and turbidimetric techniques.
Various immunoassay methods to detect and quantify valproic acid in physiological fluid test samples are known to the art. An immunoassay of valproic acid or its metabolites, must overcome several problems. One of these problems arises because valproic acid is a hapten. Hence, valproic acid is not by itself capable of inducing an immune response upon administration to an animal body, including a human body. This occurs because valproic acid is too small to be recognized by the body's immune system. However, when coupled to a carrier, such as certain proteins, the valproic acid:carrier conjugate can act as an antigen which is large enough to induce valproic acid antibody production. These antibodies can then be used in an immunoassay for valproic acid.
The valproic acid molecule is typically not coupled directly to the carrier, because the larger carrier tends to prevent a valproic acid molecule that has been directly linked to the carrier from being recognized by an animal's immune system. Generally therefore, a valproic acid derivative is prepared for coupling to the carrier. The valproic acid derivative can be a valproic acid molecule attached to one end of a spacer arm or spacer chain of a sufficient length. The free end of the spacer arm, that is the end of the spacer arm that is not attached to the valproic acid molecule, usually bears a reactive functional group. The functional group is used to link the free end of the spacer arm to a carrier, such as bovine serum albumin, to make a valproic acid conjugate.
Interposed between the valproic acid molecule and the carrier, the spacer arm thereby acts to physically space or extend the valproic acid molecule away from the carrier. The valproic acid molecule can then be recognized by an animal's immune system as an immunogen. Hence, it is highly desirable to be able to prepare a valproic acid derivative which upon linkage to a carrier results in the making of an immunoreactive valproic acid conjugate.
Besides being useful to raise antibodies against valproic acid, an immunoreactive valproic acid conjugate can also be used as a developer antigen in a competitive inhibition immunoasay for valproic acid. A developer antigen is used because the small, monovalent valproic acid molecule is unable to form a large complex or aggregate with the valproic acid antibodies. Consequently, a valproic acid derivative, such as a biotinylated valproic acid derivative, can be conjugated to a carrier, such as avidin, to make a valproic acid developer antigen. The valproic acid developer antigen is another type of immunoreactive valproic acid conjugate. The valproic acid derivative is again typically made by attaching a spacer arm to a valproic acid molecule. The interposed spacer arm permits the antivalproic acid antibodies used in an immunoassay for valproic acid to recognise and adhere to the valproic acid component of the immunoreactive valproic acid conjugate. In this manner detectable immunoprecipitates can form.
Thus, formation of either the valproic acid immunogen, or the valproic acid developer antigen for use respectively, in raising valproic acid antibodies, or as the developer reagent in a competitive inhibition immunoassay for free valproic acid in a patient test sample, first requires that a suitable valproic acid derivative be prepared for conjugation to a carrier.
It would therefore be advantageous to have a method for easily preparing significant amounts of a valproic acid derivative suitable for conjugation to a carrier. The immunoreactive valproic acid conjugates can then be used to raise anti-valproic acid antibodies or as developer antigens in a valproic acid competitive inhibition immunoassay.
Existing methods for making a valproic acid derivative having a functionalized spacer arm have considerable deficiencies and drawbacks. Thus, it is known that a valproic acid derivative amenable to conjugation to a carrier, can be made by attaching a functionalized spacer arm to the .alpha. carbon atom of the valproic acid molecule. Unfortunately, an .alpha. position derivatized valproic acid molecule cannot be reliably used to make immunoreactive valproic acid conjugate for raising antivalproic acid antibodies. Additionally, a valproic acid conjugate made from an .alpha. position derivatized valproic acid molecule tends to have highly variable immunoreactivity from one batch of such a conjugate to another.
It is also known to make a valproic acid derivative by attaching a spacer arm to a .delta. carbon atom position of a valproic acid molecule. Such .delta. position derivatized valproic acid molecules have been used to make suitable immunoreactive valproic acid conjugates. Unfortunately, significant problems exist with known methods for attaching a functionalized spacer arm to a .delta. carbon atom of the valproic acid molecule. These problems include extreme difficulty to remove the alkylating reagent once it has attached the spacer arm to a precursor valproic acid molecule. Thus, when particular aromatic reagents, such as an alkylating phthalimide is used to attach a spacer arm, the aromatic molecule can be almost intractable to removal. Removal can require high temperature and dangerous closed system conditions.
Additionally, separation of the valproic acid derivative from the reaction medium and other reaction products, and unreacted reagents, can usually be carried out only by difficult and time consuming extraction procedures.
Furthermore, existing methods for making valproic acid derivatives suitable for formation into immunoreactive valproic acid conjugates, do not use the same valproic acid precursor starting material, to make more than one type of valproic acid derivative. Different valproic acid derivatives can have different spacer arms and/or spacer arm functional groups. The ability to make a variety of valproic acid derivatives from the same valproic acid precursor starting material is highly desirable for a number of reasons. For example, different spacer arms can allow precise control of steric factors, which in turn affect binding/unbinding characteristics of the valproic acid molecule of a valproic acid conjugate with different substrates and binding partners. Additionally, the ability to vary the spacer arm functional groups can permit the valproic acid derivative to be linked to different carriers.
What is needed therefore is a method for making a valproic acid derivative that allows: (1) the alkylating reagent to be removed under mild conditions subsequent to attachment of the spacer arm; (2) easy separation of the prepared valproic acid derivative from other reactants, and by-products; (3) a plurality of valproic acid derivatives to be made from the same valproic acid precursor starting material, and; (4) preparation of valproic acid derivatives that can be used to make immunoreactive conjugates.